Coronaviruses cause a broad spectrum of respiratory disease in humans. Human coronaviruses (HCoV)-OC43 and HCoV-229E have long been known as agents of the common cold. The emergence of SARS-CoV as the etiological agent of severe acute respiratory distress syndrome in 2003 and in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), with 10% and 30% mortality, respectively, make it imperative to understand the coronavirus-host interactions that contribute to virulence. The ability of viruses to evade or antagonize type I interferon (IFN) signaling, influences viral pathogenesis. An important, but understudied aspect of IFN evasion by coronaviruses is antagonism of the potent, antiviral oligoadenylate synthetase (OAS)- ribonuclease (RNase) L pathway. Once activated by double stranded RNA, (dsRNA), OAS synthesizes 2',5'- linked oligoadenylates (2-5A) that activate RNase L. RNase L cleaves single stranded RNA leading to degradation of viral genomes, arrest of protein synthesis, and apoptosis. The group 2a Betacoronavirus, mouse hepatitis virus (MHV) accessory protein ns2 is a 2',5'-phosphodiesterase (PDE) that cleaves 2-5A thereby preventing RNase L activation. PDE activity is a critical determinant of MHV hepatovirulence in mice. Other group 2a Betacoronavirus, including HCoV-OC43, encode ns2 homologs recently confirmed as RNase L antagonists. Additionally, ORF4b of the group 2c Betacoronavirus MERS-CoV, encodes a protein predicted to have PDE activity. ORF4b homologs are encoded by all sequenced group 2c Betacoronavirus. These findings lead to the overall hypothesis that OAS-RNase L antagonism contributes to human group 2a Betacoronavirus respiratory pathogenesis and that group 2c Betacoronavirus also antagonize the OAS-RNase L pathway by PDE mediated cleavage of 2-5A, contributing to virulence. I will carry out the following aims. Aim 1 will examin the contribution of RNase L antagonism to the success of HCoV-OC43 in primary human airway cells. Metrics for analysis of antagonism will include viral titers, ribosomal RNA (rRNA) integrity and quantification of 2-5A levels. Results will be correlated with expression of OAS genes in these cells. Aim 2 will investigate the predicted PDE activity of MERS-CoV encoded ORF4b protein and two Bat-CoV homologs. PDE activity of recombinant ORF4b encoded protein will be assessed in vitro by a 2-5A cleavage assay and in transfected cells by measuring rRNA integrity and quantifying 2-5A levels by an RNase L activation assay. In addition, chimeric MHVs expressing inactive ns2 and each active ORF4b PDE will be constructed, infections performed in mice and rescue of replication and hepatitis assessed. Finally, primary human airway cells will be infected with MERS-CoV or ORF4b mutant virus to assess replication in relevant human cells. This study will contribute to the understanding of coronavirus-host interactions, in particular the importance of RNase L antagonism by human respiratory coronaviruses. Finally, viral PDEs offer a tantalizing target for antiviral therapeutics by isolating treatment to infected cells, because viral dsRNA is required for RNase L activation.